The long term goal of this project is to identify blood-brain barrier (BBB) ion transporters that mediate ischemia induced brain edema, a major cause of brain damage in stroke. During the early hours of cerebral ischemia, brain edema formation occurs in the presence of an intact BBB. In this process, BBB endothelial cells transport Na and Cl from blood into brain interstitium, with osmotically obliged water following. The specific ion transporters responsible are unknown, however BBB luminal Na and Cl transporters appear to play a key role. Much evidence indicates that hypoxia, which rapidly develops during ischemia, aglycemia occurring as glucose is depleted, and also centrally-released vasopressin are mediators of ischemia-induced brain edema formation. A novel aspect of this proposed project is the preliminary finding that a Na-K-Cl cotransporter appears to be localized in the luminal membrane of brain microvessel endothelial cells and that vasopressin, hypoxia and aglycemia stimulate activity of the cotransporter. This has led to the central hypothesis that a Na-K-Cl cotransporter, located at the luminal membrane of the BBB, is stimulated during ischemia to increase transport of Na and Cl with osmotically obliged water from blood to brain, causing edema formation. The present project has three specific aims. The first aim is to test the hypothesis that Na-K-Cl cotransport is present in luminal membranes of cerebral microvascular endothelial cells (CMEC). These studies will evaluate bovine brain microvessel luminal and abluminal membrane preparations for cotransport activity by radioisotopic flux analyses. Also, the in situ distribution of the cotransporter will be examined by immunoelectron microscopy of brain sections. The second aim is to test the hypothesis that Na-K-CI cotransport of BBB endothelial cells is stimulated by agents that mediate ischemia-induced cerebral edema. Here, the effects of hypoxia, aglycemia and vasopressin on cotransport activity will be examined in cultured human and bovine CMEC and freshly isolated bovine cerebral microvessels. The third aim is to test the hypothesis that inhibition of Na-K-Cl cotransport activity attenuates ischemia-induced brain edema. To do this, the effect of inhibiting the cotransporter on ischemia-induced changes in rat brain Na and water will be examined by nuclear magnetic resonance methods, which allow in vivo changes in brain Na and water to be followed in real time. The proposed studies should reveal whether therapeutic approaches aimed at blocking BBB Na-K-Cl cotransporter activity may be of value for attenuating ischemia-induced brain edema.